Team:Freiburg Bioware/Project/Results/Arming

Contents

Arming: Suicide Genes as GOIs. 1

Introduction. 1

Successful Assembly of Vector Plasmids Carrying Suicide Genes via Cloning. 1

Monitoring Efficient Tumor Killing by Phase-Contrast Microscopy. 4

Quantitative Analysis of Cell Death by Flow Cytometry. 6

Titrating Ganciclovir Concentrations for Efficient Cell Killing by Cytotoxicity Assays. 8

Killing Untransduced Tumor Cells via Bystander Effect 13

Conclusions. 16

References. 16

Arming: Suicide Genes as GOIs

Introduction

Gene delivery using viral vectors to specifically target tumor cells gained increasing attention in the last years being efficient in combination with suicide gene approaches (Willmon et al. 2006). Several prodrug/enzyme combinations have been reported. Two systems - ganciclovir (GCV)/herpes simplex virus thymidine kinase (HSV-TK) (Ardiani et al. 2010) and 5-fluorocytosine/cytosine deaminase (CD) (Fuchita et al. 2009a) – have been widely used and their therapeutic benefit was demonstrated in preclinical studies (Greco & Dachs 2001). Adeno-associated viruses (AAV) as delivery vectors are commonly used in suicide gene therapy. The suicide gene flanked by the inverted terminal repeats (ITRs) is encapsulated into the virus particles and delivered to the target cells where suicide gene expression is mediated by cellular proteins.

The iGEM team Freiburg_Bioware 2010 provides both the cytosine deaminase (CD, BBa_K404112) and an improved guanylate kinase - thymidine kinase fusion gene (mGMK_TK, BBa_K404113) within the Virus Construction Kit as effective suicide genes. We demonstrate efficient and specific killing of tumor cells by enzymatic cytotoxicity assays, flow cytometry, as well as phase contrast microscopy. HT1080 cancer cell lines were transduced with directed viral particles containing the suicide genes packaged into the viral capsids.

Successful Assembly of Vector Plasmids Carrying Suicide Genes via Cloning

To create the functional vector plasmids, assembly of the constructs carrying the suicide genes was performed following the BioBrick Standard Assembly. All plasmids contain the enhancer-element human beta-globin intron (BBa_K404107) and the human growth hormone terminator signal (hGH, BBa_K404108) flanked by the inverted terminal repeats (ITRs, BBa_K404100 and BBa_K404101). Assembled suicide genes are either under the control of the CMV promoter (BBa_J52034) or the tumor-specific telomerase promoter phTERT (BBa_K404106).

To enable modularization of the thymidine kinase mutants TK30 and SR39 (BBa_K404109 and BBa_K404110) according to the BioBrick standard, the fusion genes mGMK_TK30 and mGMK_SR39 (BBa_K404113 and BBa_K404315) and CD (BBa_K404112) were modified using the QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene) for deletion of iGEM RFC10 pre- and suffix restriction sites. Figure 1 demonstrates one example of successful deletion of a PstI restriction site located within the mGMK_TK30 sequence at position 3109. A point mutation was introduced replacing the nucleotide G by A, resulting in the deletion of the restriction site while maintaining the encoded amino acid glutamine. Exchange of guanine to adenine was confirmed by sequencing (Figure 2).

Furthermore, assembly of BioBrick-compatible vector plasmids was performed. An example for the last assembly step of mGMK_TK30 and hGH_rITR is shown in Figure 3. The plasmids were digested with both XbaI and PstI (New England Biolabs, Insert: BBa_K404116: hGH_rITR) or SpeI and PstI (Vector) and loaded on an agarose gel. As demonstrated in the preparative gel in Figure 3, the expected bands were detected under UV light and the extracted, subsequently purified DNA was successfully ligated and transformed into E. coli. Each assembly step for producing the BioBricks was conducted following the iGEM BioBrick standard.

Monitoring Efficient Tumor Killing by Phase-Contrast Microscopy

Tumor cells, transduced with viral particles encapsidating the effector constructs containing the mGMK_TK30 driven by the CMV promoter, were cultured both in presence and absence of ganciclovir (Roche). Morphological changes were monitored via phase-contrast microscopy for 48 hours post infection. As it can be seen in Figure 4, non-transduced tumor cells treated with ganciclovir and transduced cells without ganciclovir did not show significant cell ablation. In contrast, transduced cells expressing the guanylate kinase - thymidine kinase fusion protein showed significant cell death after incubation with ganciclovir for 48 hours post infection.

Suicide gene therapy is based on the localized conversion of non-toxic prodrugs to toxic substances (Greco & Dachs 2001), promoting cell death in the tumor tissue (Figure 5). Directed gene delivery is achieved by using recombinant viral vectors as provided by the iGEM team Freiburg_Bioware 2010 within the Virus Construction Kit.

Non-transduced cells can survive in the presence of ganciclovir since the prodrug is not toxic for these cells (Figure 4A). The demonstration that transduced cells are viable in the absence of ganciclovir confirms that cell killing is indeed induced by combination of the delivered thymidine kinase and treatment with ganciclovir. Viral particles encapsidating the suicide construct mGMK_TK30 are efficient in directed gene delivery, thus leading to cell death of transduced cells due to overexpression of mGMK_TK30 and prodrug conversion. The cell-toxic ganciclovir-triphosphate is incorporated into the nascent DNA chain leading to replication termination and finally resulting in death of dividing cells.

Quantitative Analysis of Cell Death by Flow Cytometry

Quantitative analysis of the cytotoxic effect induced by mGMK_TK30 was first conducted by flow cytometry analysis 72 hours post transduction. HT1080 cells were stained with 7-AAD and Annexin V. 7-AAD intercalates in double-stranded DNA after penetrating cell membranes of dead cells, whereas Annexin V specifically binds phosphatidylserine which is only accessible during apoptosis. Figure 6 demonstrates the relation between cell death and ganciclovir concentration.